Somatostatin (SST) is a widely distributed peptide occurring in two forms SST-14 (with 14 amino acids) and SST-28 (with 28 amino acids). It was originally isolated from the hypothalamus and characterized by Guillemin et al. (U.S. Pat. No. 3,904,594) and is described in U.S. Pat. No. 3,904,594 (Sep. 9, 1975). Somatostatin is found in the gut, pancreas, in the nervous system, in the various exocrine and endocrine glands through the body and in most organs. In normal subjects somatostatin has a broad spectrum of biological activities. It participates in a large number of biological processes where it has the role of an inhibitory factor. It inhibits the release of insulin, prolactin, glucagon, gastrin, growth hormone, thyroid stimulating hormone, secretin and cholecystokinin. (S. Reichlin: Somatostatin, N.Eng. J. Med., 309,1495 and 1556, 1983.)
The mechanism of action of somatostatin is mediated by high affinity membrane associated receptors. Five somatostatin receptors (SSTR1-5) are known. (Reisine, T; Be11,G.I; Endocrine reviews, 1995, 16, 427-42.) All five receptors are heterogeneously distributed and pharmacologically distinct. Somatostatin receptors have been found to be over-expressed in a wide range of tumors, those arising in the brain (including meningioma, astrocytoma, neuroblastoma, hypophysial adenoma, paraganglioma, Merkel cell carcinoma, and gliomas), the digestive-pancreatic tract (including insulinoma, gluconoma, AUODoma, VIPoma, and colon carcinoma), lung, thyroid, mammary gland, prostate, lymphatic system (including both Hodgkin's and non-Hodgkin's lymphomas), and ovaries.
One of the most important effects of somatostatin are its growth-inhibiting ability and its capability to influence pathological cell growth. It is well known that it exerts an inhibitory effect on the growth of cancer cells both directly and by its antagonizing action on growth factors associated with malignant growth. (A. V. Schally: Cancer. Res., 48, 6977, (1988); Taylor, et. al., Biochem., Biophys. Res. Commun., 153, 81 (1988). It has been shown by recent investigations that somatostatin and some somatostatin analogues are capable of activating the tyrosine phosphatase enzyme which antagonizes the effect of tyrosine kinases playing a very important role in the tumorous transformation (A. V. Schally: Cancer Res. 48, 6977 (1988)). The importance of tyrosine kinases is supported by the fact that the majority of oncogenes code for tyrosine kinase and the major part of growth factor receptors is tyrosine kinase (Yarden et al.: Ann. Rev. Biochem. 57, 443 (1989)).
Native somatostatin has a very short or transient effect in vivo since it is rapidly inactivated by endo- and exopeptidases. A large number of novel analogues have been synthesized in order to increase its plasma half life and biological activity. Most of the active analogues contain a disulphide bond and a peptide chain shorter than the original one. The first cyclic hexapeptide showing the whole effects of somatostatin was synthesized by Veber et al. (Nature, 292, 55 (1981)). Newer and more effective cyclic hexa- and octapeptides have been synthesized which possess the whole spectrum of effects of somatostatin (Veber et al.; Life Sci. 34, 1371 (1984); Murphy et al.; Biochem. Biophys. Res. Commun. 132, 922 (1985); Cai et al.; Proc. Natl. Acad. Sci. USA 83, 1896 (1986)).
In spite of the high rates of over expression of somatostatin receptors on a variety of tumors, somatostatin analogues have not gained widespread clinical application for the control of cancer. Their current clinical application is primarily in the control of symptoms associated with metastatic carcinoid or VIP-secreting tumors. The somatostatin analogues have a wide therapeutic index and seem to be free of major side effects. Most of the side effects are gastrointestinal in nature and include minor nausea, bloating, diarrhea, constipation, or steatorrhea. Part of the reason for the restricted clinical use may be due to the need for long-term maintenance therapy, the consequent high cost of such therapy, and the variable effects observed in clinical settings.
Some somatostatin analogues, preparation of such analogues, and uses for such analogues are known in the prior art. Such analogues are used in the treatment of certain cancers and other conditions. One commercially available product, octreotide, D-Phe-Cys-Phe-D-Trp-Lys-Thr-Cys-Thr-of (SEQ ID NO.: 1) (disulphide bridge between the Cys residue), manufactured by Sandoz, and sold under the trade name Sandostatin, is being used clinically to inhibit tumor growth and as a diagnostic agent to detect somatostain receptor expressing tumors. Of the five receptor sub-types, octreotide and other clinically used somatostatin analogs interact significantly with three of the receptor sub-types, SSTR2, SSTR3 and SSTRS. SSTR2 and SSTRS have recently been reported to mediate anti-proliferative effects of somatostatin on tumor cell growth, and may therefore mediate the effects of octreotide in humans.
A wide variety of somatostatin analogues have been developed. These include RC-160, a potent somatostatin analogue originally synthesized by a team at Tulane University headed by Andrew V. Schally (Cai R. Z., Szoke B., Lu E., Fu D., Redding T. W. and Schally A. V.: Synthesis and biological activity of highly potent octapeptide analogues of somatostatin. Proc Natl Acad Sci USA, 83:1896-1900, 1986). In recent studies conducted by Schally, among others, the effectiveness of RC-160 in inhibiting the growth of humor glioblastomas in vitro and in vivo has been demonstrated (Pinski J, Schally A V, Halmos G, Szepeshazi K and Groot K: Somatostatin analogues and bombesin/gastrin-releasing peptide antagonist RC-3095 inhibit the growth of human glioblastomas in vitro and in vivo. Cancer Res 54:5895-5901, 1994).
Recent patents that describe somatostatin analogs for treatment of cancer are following:
U.S. Pat. No. 6,025,372 (February 2000) PA1 WO 0006185A2 (February 2000) PA1 WO 9922735A1 (May 1999) PA1 WO 9845285A1 (October 1998) PA1 WO 9844921A1 (October 1998) PA1 WO 9844922A1 (October 1998) PA1 U.S. Pat. No. 5,753,618 (May 1998) PA1 U.S. Pat. No. 5,597,894 (January 1997) PA1 EP 0344297E 1 (May 1994) PA1 JP 5124979A (May 1993) PA1 U.S. Pat. No. 4,904,642 (February 1990) PA1 BOP: Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexofluorosphospate PA1 PyBOP: Benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexofluorophospate PA1 HBTU: O-Benzotriazole-N,N,N',N'-tetramethyl-uronium-hexofluoro-phosphate PA1 TBTU: 2-(1H-Benzotriazole-1yl)-1,1,3,3-tetramethyluronium tetrafluoroborate PA1 HOBt: 1-Hydroxy Benzotriazole PA1 DCC: Dicyclohexyl carbodiimide PA1 DIPCDI: Diisopropyl carbodiimide PA1 DIEA: Diisopropyl ethylamine PA1 DMF: Dimethyl formamide PA1 DCM: Dichloromethane PA1 NMP: N-Methyl-2-pyrrolidinone PA1 TFA: trifluoroacetic acid PA1 Orn=Ornithine PA1 Pen=Penicillamine PA1 Aib=.alpha.-Aminoisobutyric acid PA1 Deg=.alpha., .alpha.-Di-ethyl glycine PA1 Dpg=.alpha., .alpha.-Di-n-propyl glycine PA1 Ac5c=1-Aminocyclopentane carboxylic acid PA1 X is Acetyl or straight, branched, or cyclic alkanoyl group from 3 to 18 carbon atoms, or is deleted; PA1 A1=Orn or Lys; PA1 A2=Aib, Deg, Dpg or Ac5c; PA1 A3=Pen or Cys; or a hydrolyzable carboxy protecting group; or a pharmaceutically acceptable salt of the peptide.
The aim of the present invention is to synthesize novel somatostatin analogs showing a more advantageous and more selective biological action in comparison to that of known compounds. The invention is based on the use of .alpha., .alpha.-dialkylated amino acids in the octapeptide analog of somatostatin at position 6. These amino acids are known for inducing conformational constraint. The design of conformationally constrained bioactive peptide derivatives has been one of the most widely used approaches for the development of peptide-based therapeutic agents. Non-standard amino acids with strong conformational preferences may be used to direct the course of polypeptide chain folding, by imposing local stereochemical constraints, in de novo approaches to peptide design. The conformational characteristics of .alpha., .alpha.-dialkylated amino acids have been well studied. The incorporation of these amino acids restricts the rotation of .PHI., .PSI. angles, within the molecule, thereby stabilizing a desired peptide conformation. The prototypic member of .alpha., .alpha.-dialkylated aminoacids, .alpha.-aminoisobutyric acid (Aib) or .alpha., .alpha.-dimethylglycine has been shown to induce .beta.-turn or helical conformation when incorporated in a peptide sequence (Prasad, B. V. V. and Balaram, P. CRC Crit Rev. Biochem. 16,307-347 (1984), Karle, L L. and Balaram, P. Biochemistry 29, 6747-6756, (1990)). The conformational properties of the higher homologs of .alpha., .alpha.-dialkylated amino acids such as di-ethylglycine (Deg), di-n-propylglycine (Dpg) and di-n-butylglycine (Dbg) as well as the cyclic side chain analogs of .alpha., .alpha.-dialkylated amino acids such as 1-aminocyclopentane carboxylic acid (Ac5c), 1-aminocyclohexane carboxylic acid (Ac6c), 1-aminocycloheptane carboxylic acid (Ac7c) and 1-aminocyclooctane carboxylic acid (Ac8c) have also been shown to induce folded conformation (Prasad, S. et al., Biopolymers 35, 11-20 (1995); Karle, 30 LL. et al., J. Amer. Chem.Soc. 117, 9632-9637(1995)). .alpha., .alpha.-Dialkylated amino acids have been used in the design of highly potent chemotactic peptide analogs (Prasad, S. et al., Int. J. Peptide Protein Res. 48, 312-318, (1996)).
The present invention exploits the conformational properties of .alpha., .alpha.-dialkylated amino acids for the design of biologically active peptide derivatives, taking somatostatin as the model system under consideration. The invention is directed to somatostatin analogs containing .alpha., .alpha.-dialkylated amino acids. A further object of the invention is the synthesis of somatostatin analogs containing .alpha., .alpha.-dialkylated amino acids. The inventors have also synthesized peptide derivatives having N-terminal alkanoyl groups of from C2 to C18 carbon atoms, which retain anticancer activity. A still further object of the invention is the preparation of the analogs by solid phase peptide synthesis methodology. It has been shown that lipophilazation of bioactive peptides improves their stability, bioavailability and the ability to permeate biomembranes (Dasgupta, P. et al.; 1999, Pharmaceutical Res. 16, 1047-1053; Gozes, I. et al., 1996, Proc. Natl. Acad. Sci.USA, 93, 427-432).
Throughout the specification and claims, the following abbreviations are used with following meanings:
In formula (I) below and throughout the specification, and claims the amino acids residues are designated by their standard abbreviations. Amino acids denote L-configuration unless otherwise indicated by D or DL appearing before the symbol and separated from it by hyphen.
The following abbreviations are used for noncommon amino acids: